The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the present invention.
Since dopamine (DA) was identified as a brain neurotransmitter 50 years ago (Carlsson et al., 1957), numerous scientists demonstrated its critical role in normal as well as in diseased brains. The majority of DA neurons reside in the ventral mesencephanlon, forming midbrain DA (mDA) neurons. They critically control voluntary movement, reward, and mood-related behaviors, and their degeneration/dysfunction is associated with major brain disorders such as Parkinson's disease (PD) and schizophrenia. Thus, purification and characterization of expandable mDA progenitor cells is crucial for the design of effective therapeutic approaches for these diseases as well as to provide an in-depth understanding of mDA neuron development and biology. Recent developments in pluripotent stem cell technology such as embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) promise an unlimited quantity of differentiated cells for such purposes, only if there is a reliable method by which specific progenies can be isolated/purified from heterogeneous population of differentiated cells.
Despite many studies from different labs, no single marker that can purify mDA neural progenitor cells (NP cells) has yet been found. However, based on the knowledge gained from developmental studies of mDA neurons in this and other laboratories, mDA NPs can be identified and purified. Dysfunction of mDA neurons has been implicated in various brain diseases such as neurodegenerative and psychiatric disorders. In particular, the selective degeneration of mDA neurons causes PD, one of the most frequent neurodegenerative disorders. PD is often diagnosed when more than 70-80% of DA nerve terminals in the striatum have been degenerated (Agid, 1991). Thus, the need for reconstructive therapies to treat PD led to the development of fetal cell transplantation therapies (Lindvall and Bjorklund, 2004).
Whereas fetal DA cell transplantation showed the proof-of-principle of cell-based therapy of PD, its use is limited by the lack of standardized fetal cells and ethical controversies. Alternatively, mDA neurons can be derived from ESCs as an unlimited cell source (Bjorklund et al., 2002; Chung et al., 2002; Kawasaki et al., 2000; Kim et al., 2002; Perrier et al., 2004; Roy et al., 2006), but ES-derived progenies are heterogeneous, thus rendering control of their function after transplantation difficult, which is one of the major obstacles for clinical application of ESCs. ESC-derived cells often contain more immature cells and even residual pluripotent cells that can form tumors (Chung et al., 2006a; Roy et al., 2006; Schulz et al., 2004; Zeng et al., 2004). In addition, the lack of a standardized cell source and unfavorable cell composition (e.g. too many serotonergic neurons) can result in complications such as graft-induced dyskinesia after transplantation (Lindvall and Kokaia, 2009; Politis et al., 2010).
Thus, purification of desired cell types from differentiated ESC prior to transplantation is critical for the safety and efficient function of the grafts. Furthermore, isolation of functionally verified mDA cells from ESC-derived progenies can provide valuable resources to study the biology of mDA NP cells and mDA neurons, which is crucial to further understanding of the etiology of PD and the design of effective therapeutic approaches. What is more, it will also serve as a bioassay and drug screening tools, thus facilitating a pharmacological intervention for the treatment of PD. Recently developed iPSC technology (Takahashi and Yamanaka, 2006) offers the possibility to generate disease- or patient-specific stem cells, which could provide a way to model a disease in a dish or to avoid immune rejection caused by non-autologous cell therapy. However, to realize the full translational potential of these pluripotent cells (e.g., ESCs and iPSCs), it is critical to develop reliable and optimal methods to identify and purify specific cell populations. Furthermore, given the extreme vulnerability and poor survival of terminally differentiated neurons in vitro and in vivo, it is important to identify and isolate specific neural progenitor cells that are expandable and able to better survive.